The intracellular infectious agent Toxoplasma gondii is a member of the phylum Apicomplexa, which includes the organisms responsible for malaria and coccidiosis. Although most Toxoplasma infections are asymptomatic, they can lead to severe disease and even death in utero and in individuals immunocompromised by AIDS or cancer. Some of the devastating effects of a Toxoplasma infection are a direct consequence of its lytic cycle, which consists of attachment to the host cell, invasion, intracellular replication and egress. The process of egress involves fluctuation in intracellular [Ca+2], morphological rearrangements and the permeabilization of the host cell. The specific genes and cues involved in this rapid and active exit from the host cell are not known. Up to now, the genetic dissection of egress has concentrated on characterizing mutants with a delay in artificially induced egress. Since none of the mutants analyzed so far show obvious defects in natural egress, new approaches are needed to directly identify the genes involved in egress. Selecting, as opposed to screening, for parasites with a delay in egress is complicated by the lack of synchrony of this process and the fact that egress mutant parasites will likely have a growth disadvantage and thus would be outgrown before any expansion through a selection can take place. On the other hand, mutants with both growth and egress defects can be isolated by screening through independently established and maintained parasite clones. Accordingly, a library of 10,000 insertional and chemical mutants will be created and mutants with deficient egress will be isolated. The phenotypic and molecular analysis of these mutants will allow for the identification of the genes essential for Toxoplasma egress and elucidate the mechanisms and pathways involved in this critical step of the life cycle of this human pathogen. Furthermore, these studies are likely to shed light to important cell biology questions such as organellar secretion, ion homeostasis and cytoskeleton rearrangement. [unreadable] [unreadable]